Jacobs:Protocol RNA Sample Loading Buffer

Overview

RNA sample loading buffer is formulated for use in RNA sample preparation for denaturing formaldehydeagarose gel electrophoresis. The solution contains tracking dyes and ethidium bromide, so gel loading buffer and ethidium bromide staining are not required.

Materials

• Composition:
  • 62.5 % deionized formamide
  • 1.14 M formaldehyde
  • 1.25X MOPS-EDTA-sodium acetate buffer (Product No. M 5755, diluted 1:8)
  • 200 μg/ml bromphenol blue
  • 200 μg/ml xylene cyanole
  • 50 μg/ml ethidium bromide (See note below)
• Suitable for use in formaldehyde-agarose gel electrophoresis of RNA.
  • RNase: None detected
  • Store at 0 to −20 °C

Note: This solution contains ethidium bromide that can interfere with transfer of RNA to nylon or nitrocellulose membranes.

Procedure

1. Prepare Standards ( 2 fold dilutions): 2 µg/ µl, 1 µg/ µl, 0.5 µg/ µl, 0.25 µg/ µl, 0.125 µg/ µl
   1. Label 5 microcentrifuge tubes 1-5 (1= highest concentration, 5= lowest concentration)
   2. In tube #1, put in 120 µl of BSA, in all other tubes put in 60 µl of RIPA lysis Buffer (you always want to use the same dilutant material as what you used to isolate your protein)
   3. Pipette 60 µl of BSA from tube #1 into #2. Pipette up and down a dozen times or until you think it is properly mixed. Then take 60 µl from tube #2 and put it in tube #3 and pipette up and down. Continue this process through tube #5 (this will leave tube #5 with 120 µl)
2. Prepare BCA Working Reagent
   1. For the total volume of working reagent calculate:
      • (# standards (in our case 5) and samples (30 in our case))*(# replicates (2))*(volume of working solution per sample (200 µl))
   2. To prepare working solution mix 50 parts Reagent A with 1 part Reagent B (ie. 50 ml Reagent A plus 1 ml Reagent B)
3. Prepare your samples
   1. Make three concentrations of your samples in three new microcentrifuge tubes labeled S1, S2, S3(to ensure you get within the range of 0.125-2 µl)
      1. Put 60 µl of your sample in tube S1
      2. Make a 1:2 dilution (30 µl sample + 30 µl RIPA buffer) in S2
      3. Make a 1:10 dilution ( 10 µl sample + 90 µl RIPA buffer) in S3
4. Prepare your Microplate
   1. Pipette 25 µl of each standard or unknown sample replicate into the designated microplate well
   2. Add 200 µl of the WR to each well and mix plate thoroughly on a plate shaker for 30 seconds
   3. Incubate plate at 37C for 30 minutes
   4. Remove plate and measure the absorbance at 562 nm on a plate reader
   5. Subtract the average 562 nm absorbance measurement of the Blank standard replicates from the 562 absorbance of al the individual standard and unknown
   6. Prepare a standard curve by plotting the average Blank=corrected 562 nm measurement for each BSA standard vs. its concentration in µg/µl. Use the standard curve to determine the protein concentration of each unknown sample

Notes

Used at Stanford for Tissue Engineering Lab Course (ME385B and 2007 Winter/Summer TC Workshops

Contact

• Originally prepared by CRJ-EJC 1/3/06

or instead, discuss this protocol.